Protoporphyria is characterized biochemically by elevated protoporphyrin levels and clinically by photosensitivity and hepatobiliary disease. Protoporphyria is generally inherited as an autosomal dominant disease. The enzymatic activity of ferrochelatase, which catalyzes the last step in the heme biosynthetic pathway, is reduced to 15% - 50% of normal in all tissues of patients with protoporphyria. The molecular mechanism by which ferrochelatase activity is reduced below the 50% of normal predicted for a null allele in an autosomal dominant disease is unknown, but a hypothesis is proposed in which the mutant ferrochelatase protein functions as a dominant negative mutation to inhibit the activity of the wild-type ferrochelatase protein. We have recently cloned a murine ferrochelatase cDNA, a human ferrochelatase cDNA, and the entire human ferrochelatase gene. Using the polymerase chain reaction, we have cloned multiple copies of ferrochelatase cDNAs from well characterized patients with protoporphyria. We have detected a point mutation in the coding region of the ferrochelatase where a phenylalanine is converted to a serine (F417S) in the protein product. Expression of recombinant ferrochelatase proteins in E coli demonstrate a marked deficiency in activity of the mutant ferrochelatase protein. Expression of the wild- type ferrochelatase cDNA in stably transfected human 293 cells results in increased ferrochelatase activity, while expression of the mutant ferrochelatase cDNA does not increase activity. The specific aims of this proposal are: 1. To characterize the transcriptional regulation of the human ferrochelatase gene in erythroid and non-erythroid cells. 2. To determine mutations in the ferrochelatase gene and correlate mutations with disease activity in patients with protoporphyria. 3. To assess the functional significance of structural or regulatory mutations in the ferrochelatase genes of patients with protoporphyria. 4. To develop a transgenic mouse model of protoporphyria.